Central Hypothesis: The Hepatitis C Virus (HCV) E2 glycoprotein is a novel kinase that initiates signal transduction mechanisms modulating the following pathways: HCV association with cellular receptors (CD-81, SR-B1, LDL R) and cellular tight junction proteins (Claudin 1, and occludin) that are indispensible for initial viral binding to the cellular membrane and subsequent membrane fusion mechanisms that lead to viral entry. Clathrin-mediated endocytosis (CME), through site-specific phosphorylations of <2(AP50), a subunit of the heterodimeric cargo adaptor AP2, a key regulator of clathrin-mediated receptor endocytosis and other cellular alternative CME adaptor proteins including NUMB, ARH, and Dab2 that are known to be regulated through phosphorylation at the postulated E2 phosphorylation consensus site. HCV E2 kinase activity may also affect other critical adaptor proteins including; HIP1, 2-arrestin, epsin, CALM, and PD2K1 by association/phosphorylation. Trans Golgi network (TGN) sorting and exocytosis of the viral particle, through site-specific phosphorylations of <1 a subunit of the heterodimeric cargo adaptor AP1, a key regulator of TGN sorting and other cellular TGN proteins including the Golgi-localized, 3-adaptin homologue and ARF- binding proteins (GGA1-3), COPI, COPII, Sar and ARF. Hepatocyte proliferation and liver carcinogenesis through the activation of host cellular signal transduction pathways, such as the Akt pathway. In pilot transfection studies and in vitro kinase assays I have obtained compelling data suggesting that E2 is a novel member of the actin-regulating kinase family (Ark/Prk kinases) that associates physically with, and phosphorylates AP50 on its phospho-acceptor Thr156, a key step for clathrin-mediated endocytosis. Also, we have shown that E2 is associated with AP50 (<2) in livers from HCV-infected patients, and that AP50 is phosphorylated on Thr156 to a much greater extent in these livers. In preliminary studies, we have also found that E2, in the absence of extracellular growth factors, increases the expression and activities of PIP2, PI3K, PDK1 and Akt. This signaling cascade promotes proliferation. Moreover, HCV E2 markedly stimulates hepatocyte DNA replication to an even greater extent than classic tumor promoters TGF1 and EGF. This proposal will study the signal transduction mechanisms by which HCV associates with CD81, SR- B1(9), LDL R(10), cellular receptors and tight junction proteins, claudin 1 and occludin, and phosphorylates <2 (AP2), NUMB, ARH, Dab2, GULP/CED-6 , HIP1, 2-arrestin, CALM, and PD2K1 thereby modulating initial attachment, membrane-fusion associated events and CME resulting in viral entry and endocytosis. I will also investigate the analogous TGN sorting signals that lead to viral exocytosis. These are controlled by AP1 (<1) phosphorylation (14) and so like the AP2 (<2) phosphorylation during endocytosis may also be governed by E2. This proposal will also focus on how E2 induces the Akt pathway and increases hepatocyte proliferation, thereby facilitating liver carcinogenesis.